Methods for preparation and use of 1A,24(S)-dihydroxy vitamin D2

ABSTRACT

1 alpha ,24(S)-Dihydroxy vitamin D2 which is useful as an active compound of pharmaceutical compositions for the treatment of disorders of calcium metabolism and for various skin disorders. The invention also includes preparation of synthetic 1 alpha ,24(S)-dihydroxy vitamin D2 starting from ergosterol which is converted in six steps to 24-hydroxyergosterol. 24-Hydroxyergosterol is irradiated and thermally converted to 24-hydroxy vitamin D2 which is converted in six steps to 1 alpha ,24(S)-dihydroxy vitamin D2. The syntheses also produced novel intermediates.

This is a division of application Ser. No. 08/275,641 filed Jul. 14,1994, which is a continuation of application Ser. No. 07/940,246, filedAug. 28, 1992, now abandoned, which is a continuation-in-part ofapplication Ser. No. 07/637,867, filed Jan. 8, 1991, now abandoned, anda Section 371 of PCT/US92/00313, filed Jan. 7, 1992, and whichdesignated the U.S.

TECHNICAL FIELD

This invention relates to biologically active vitamin D₂ compounds. Morespecifically, this invention relates to the hormonally active, naturalmetabolite 1α,24(S)-dihydroxy vitamin D₂ and to methods of preparingthis metabolite and the nonbiological epimer 1α,24(R)-dihydroxy vitaminD₂. This invention also relates to a pharmaceutical composition whichincludes a pharmaceutically effective amount of 1α,24(S)-dihydroxyvitamin D₂, and to a method of controlling abnormal calcium metabolismby administering a pharmaceutically effective amount of the compound.

BACKGROUND OF THE INVENTION

Vitamin D and its active metabolites are known to be important inregulating calcium metabolism in animals and humans. The naturallyoccurring form of vitamin D in animals and humans is vitamin D₃. It hasbeen shown that in animals, including humans, vitamin D₃ is activated bybeing hydroxylated in the C₂₅ position in the liver, followed by1α-hydroxylation in the kidney to produce the hormone 1α,25-dihydroxyvitamin D₃ "1α,25-(OH)₂ D₃ "!. See, U.S. Pat. No. 3,880,894. The majorphysiological pathway for catabolism of the vitamin D₃ metabolites,25-hydroxy vitamin D₃ and 1α,25-(OH)₂ D₃, is initiated by C₂₄-oxidation. Holick, M. F., Kleiner-Bossallier, A., Schnoes, H. K.,Kasten, P. M., Boyle, I. T., and DeLuca, H. F., J. Biol. Chem., 248,6691-6696 (1973).

Vitamin D₂ is the major, naturally occurring form of vitamin D found inplants. Vitamin D₂ differs structurally from vitamin D₃ in that vitaminD₂ has a methyl group at C₂₄ and has a double bond between C₂₂ and C₂₃.

Shortly after their discovery, it seemed apparent that vitamin D₃ andvitamin D₂ had similar, if not equivalent, biological activity. It hasalso been commonly believed that the metabolism (i.e., the activationand catabolism) of vitamin D₂ was the same as for vitamin D₃. See,Harrison's Principles of Internal Medicine: Part Seven, "Disorders ofBone and Mineral Metabolism: Chap. 35," in E. Braunwald, K. J.Isselbacher, R. G. Petersdorf, J. D. Wilson, J. B. Martin and H. S.Fauci (eds.), Calcium, Phosphorus and Bone Metabolism: CalciumRegulating Hormones, McGraw-Hill, New York, pp. 1860-1865. In thisregard, the active form of vitamin D₂ is believed to be 1α,25-dihydroxyvitamin D₂ "1α,25-(OH)₂ D₂ "!. Further, 24-hydroxy derivatives of25-hydroxy vitamin D₂ and 1α,25-(OH)₂ D₂, that is, 24,25-dihydroxyvitamin D₂ and 1α,24,25-trihydroxy vitamin D₂, are known, suggestingthat catabolism of vitamin D₂, like vitamin D₃, proceeds through thesame C₂₄ oxidation step. Jones, G., Rosenthal, D., Segev, D., Mazur, Y.,Frolow, F., Halfon, Y., Robinavich, D. and Shakked, Z., Biochemistry,18:1094-1101 (1979).

It has recently been found, however, that an active analogue of vitaminD₂, 1α-hydroxy vitamin D₂ "1α-(OH)D₂ "! has pharmacological propertiesdistinctly different than those exhibited by its vitamin D₃ counterpart,1α-hydroxy vitamin D₃ "1α-(OH)D₃ "!. U.S. Pat. No. 5,104,864 disclosesthat 1α-(OH)D₂ will reverse the loss of bone mass in human osteoporoticpatients when administered at dosages of 2.0 μg/day or higher. Becauseof toxicity, dosage levels of 2.0 μg/day or greater are not safelyobtained with 1α-(OH) D₃.

Such distinct pharmacological properties may be explained fully, or inpart, by the present inventors' discovery that pharmacological dosagesof 1α-(OH)D₂ administered to humans are metabolized in part tobiologically active 1α,24(S)-dihydroxy vitamin D₂ "1α,24(S)-(OH)₂ D₂ "!.As explained in more detail below, the hydroxylation at the carbon-24position of the 1-hydroxylated vitamin D₂ molecule, represents anactivation pathway peculiar to the vitamin D₂ molecule.

While 1α,24(S)-dihydroxy vitamin D₃ and 1α,24(R)-dihydroxy vitamin D₃"1α,24(R/S)-(OH)₂ D₃ "! have been chemically synthesized (U.S. Pat. No.4,022,891) it has not been demonstrated that either is a naturalcompound found in biological systems. Furthermore, the present inventorshave discovered that 1α,24(S)-(OH)₂ D₂ has distinctly differentbiological activity from that exhibited by 1α,24(R/S)-(OH)₂ D₃. Forexample, Ishizuka et al. have found that 1α,24(R)-(OH)₂ D₃ binds the1,25-(OH)₂ D₃ receptor site more tightly than does 1,25-(OH)₂ D₃ itself.Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids,37:1,33-42 (1981); Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto,Y., Steroids, 39:1,53-62 (1982). Using a similar assay, the presentinventors have discovered that the 1α,24(S)-(OH)₂ D₂ is two-fold lesscompetitive in binding the 1,25-(OH)₂ D₃ receptor site than is1,25-(OH)₂ D₃. The present inventors have also found that 1α,24(S)-(OH)₂D₂ shows a relatively poor binding affinity for the vitamin D serumbinding protein which is evidence of a rather short half life indicativeof low toxicity.

The present inventors have demonstrated the presence of circulating1α,24(S)-(OH)₂ D₂ in humans administered 1α-(OH)D₂. This indicates thatin animals and man, vitamin D₂ is naturally metabolized to both1α,25-(OH)₂ D₂ and 1α,24(S)-(OH)₂ D₂. The relative ratios of the twovitamin D₂ hormones appear to vary according to the precursor and theamount of precursor presented to the C₂₄ pathway. Thus it appears thatas dosages of 1α-(OH)D₂ are increased, the ratio of 1α,24(S)-(OH)₂ D₂ to1α,25-(OH)₂ D₂ increases.

These results which are presented in more detail below, indicate that1α,24(S)-(OH)₂ D₂ has the desirable characteristic of high biologicalactivity with low toxicity. The fact that 1α,24(S)-(OH)₂ D₂ is asignificant metabolite when pharmacological levels of 1α-(OH)D₂ areadministered indicates that 1α,24(S)-(OH)₂ D₂ may be mediating thedesirable pharmacological effects of 1α-(OH)D₂ and is a usefultherapeutic drug for treating various types of disorders involvingcalcium metabolism.

SUMMARY OF THE INVENTION

The invention provides synthetic 1α,24(S)-(OH)₂ D₂ which is abiologically produced active form of vitamin D₂. The biological form mayalso be referred to as 1α,24(S)-dihydroxy ergocalciferol and isrepresented by the structure given hereinafter. The biological form ofthe compound has potent biological activity and rapid systemicclearance, indicating low toxicity.

The invention also encompasses a novel method of producing1α,24(S)-dihydroxy vitamin D₂ which entails using ergosterol as astarting material, forming 24-hydroxy vitamin D₂ and then,1α-hydroxlyating the 24-hydroxy compounds and separating the1α,24(S)-dihydroxy vitamin D₂ epimer from the 1α,24(R)-dihydroxy vitaminD₂ epimer. In the course of this synthesis, novel intermediates are alsoproduced.

The compound of the invention is useful in the treatment of variousdiseases characterized by vitamin D deficiency and various bonedepletive disorders, in particular, treatment without the concomitantincidence of hypercalcemia or hypercalciuria. The compound of theinvention is advantageously used as an active ingredient ofpharmaceutical compositions for vitamin D deficiency diseases, forreversing or preventing the loss of bone mass or bone mineral content inpersons predisposed to developing such loss, and for stabilizing bonedensity in persons suffering from renal osteodystrophy.

The compound of the invention is also useful as a topical agent fortreatment of certain skin disorders. The compound of the invention isadvantageously used as an active ingredient for topical compositionswhich may also include other agents capable of ameloriating skindisorders.

Other advantages and a better appreciation of the specific adaptations,compositional variations, and physical and chemical attributes of thepresent invention will be gained upon an examination of the followingdetailed description of the invention, taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will hereinafter be described in conjunction withthe appended drawings, wherein like designations refer to like elementsthroughout and in which:

FIG. 1 illustrates preparative steps for the synthesis of 24-hydroxyvitamin D₂ ;

FIG. 2 illustrates preparative steps for the synthesis of1α,24(S)-dihydroxy vitamin D₂ starting with 24-hydroxy vitamin D₂ ;

FIG. 3 is a reverse phase high pressure liquid chromatography profile ofbiological 1α,24-dihydroxy vitamin D₂ and the R and S epimers ofsynthetic 1α,24-dihydroxy vitamin D₂ ; and

FIG. 4 is a graph illustrating the relative binding affinities of1α,24(S)-(OH)₂ D₂ and 1α,24(R)-(OH)₂ D₂.

DETAILED DESCRIPTION

The present invention provides synthetic 1α,24(S)-dihydroxy vitamin D₂1α,24(S)-(OH)₂ -D₂ !.

As used herein, the terms "biological activity", "biologically active","bioactive", or "biopotent" are meant to refer to biochemical propertiesof compounds such as affecting metabolism, e.g., affecting serum calciumconcentration, or binding to an appropriate receptor protein, e.g.,binding to vitamin D receptor protein. The term "substantially pure" inreference to compounds or substances means a purity of at least 90%.

In one of its aspects, the invention encompasses the biologically activecompound of the formula (I): ##STR1## i.e., 1α,24(S)-dihydroxy vitaminD₂.

In another aspect, the invention involves the preparation of1α,24(S)-dihydroxy vitamin D₂. Synthesis of 1α,24(S)-dihydroxy vitaminD₂ is accomplished according to the schema presented in FIGS. 1 and 2.Hereinafter when reference is made to a 24-hydroxy compound, unlessspecified, it will be presumed that the compound is an epimeric mixtureof the R and S forms. As seen in FIG. 1, the synthesis uses ergosterolas the starting material. Ergosterol is converted to24-hydroxyergosterol (5,7,22 ergostatriene-3β,24-diol (7)) by afive-step process. The 24-hydroxy ergosterol is then irradiated andthermally converted by methods well known in the art to yield 24-hydroxyvitamin D₂. As seen in FIG. 2, 24-hydroxy vitamin D₂ is thenhydroxylated in a five-step process to yield 1α,24-dihydroxy vitamin D₂,using a procedure similar to that described by Paaren, et al., J. Org.Chem., vol. 45, p. 3253 (1980), from which the epimers are separated.

Specifically, ergosterol is acetylated to form the 3β-acetate (2). Anadduct (3) is then formed with the B-ring of the ergosterol structure byreaction of the 3β-acetate with a triazoline dione. The adduct (3) isthen ozonated to truncate the side chain to form a C-21 aldehyde (4).The side chain is reestablished by reaction of the resulting aldehydewith the appropriate keto-compound to yield the 24-enone (5). The enoneis then converted to the 24-methyl, 3β,24-dihydroxy adduct (6). Thisadduct is then reacted with a lithium aluminum hydride to deprotect theadduct and yield 24-hydroxy ergosterol (7). The 24-hydroxy ergosterol isthen irradiated and thermally treated to form 24-hydroxy vitamin D₂. The24-hydroxy vitamin D₂ is then tosylated to yield 3β-tosylate of the24-hydroxy vitamin D₂. The tosylate is displaced by solvolysis to yieldthe 6-methoxy-24-hydroxy-3,5-cyclo vitamin D₂. The cyclovitamin D₂ issubjected to allylic oxidation to form the 1α,24-dihydroxy cyclovitaminderivative. The 1α,24-dihydroxy cyclovitamin derivative is sequentiallysolvolyzed and subjected to a Diels-Alder type reaction which removesthe 6-methoxy group and separates the 1α,24-dihydroxy vitamin D₂ (5,6cis) from the 5,6 trans 1α,24-dihydroxy vitamin D₂.

The 1α,24-(OH)₂ D₂ is subjected to reverse phase high pressure liquidchromatography to separate the two epimers and recover the epimeric formof the invention, 1α,24(S)-(OH)₂ D₂.

The compound of the invention is applicable to various clinical andveterinary fields, and is particularly useful for the treatment ofabnormal metabolism of calcium and phosphorus. Specifically,1α,24(S)-dihydroxy vitamin D₂ is intended to be used, for example, tostimulate osteoblastic activity, as measured by serum levels ofosteocalcin. Osteocalcin is one of the major proteins in the bonematrix. The 1α,24(S)-dihydroxy vitamin D₂ binds to the vitamin D serumbinding protein more weakly than does 1,25-(OH)₂ D₃, indicative of rapidclearance and low toxicity, which enhances its pharmaceuticalproperties.

In a further aspect, the invention entails a method of controllingcalcium metabolism, such as for treating abnormal calcium metabolismcaused, e.g., by liver failure, renal failure, gastrointestinal failure,etc. The 1α,24(S)-dihydroxy vitamin D₂ can be used to treatprophylactically or therapeutically vitamin D deficiency diseases andrelated diseases, for example, renal osteodystrophy, steatorrhea,anticonvulsant osteomalacia, hypophosphatemic vitamin D-resistantrickets, osteoporosis, including postmenopausal osteoporosis, senileosteoporosis, steroid-induced osteoporosis, and other disease statescharacteristic of loss of bone mass, pseudodeficiency (vitaminD-dependent) rickets, nutritional and malabsorptive rickets,osteomalacia and osteopenias secondary to hypoparathyroidism,post-surgical hypoparathyroidism, idiopathic hypoparathyroidism,pseudohypoparathyroidism, and alcoholism.

1α,24(S)-Dihydroxy vitamin D₂ is also of value for the treatment ofhyperproliferative skin disorders such as psoriasis, eczema, lack ofadequate skin firmness, dermal hydration, and sebum secretion, and isvaluable for the treatment of breast and colon cancer.

1α,24(S)-Dihydroxy vitamin D₂ is useful as an active compound inpharmaceutical compositions having reduced side effects and low toxicityas compared with the known analogs of active forms of vitamin D₃, whenapplied, for example, to diseases induced by abnormal metabolism ofcalcium. These pharmaceutical compositions constitute another aspect ofthe invention.

The pharmacologically active compound of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, e.g., mammals including humans.For example, the 1α,24(S)-dihydroxy vitamin D₂ can be employed inadmixtures with conventional excipients, e.g., pharmaceuticallyacceptable carrier substances suitable for enteral (e.g., oral),parenteral, or topical application which do not deleteriously react withthe active compound.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils(e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil,coconut oil), mineral oil, fish liver oils, oily esters such asPolysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g.,lactose, amylose or starch), magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc.

The pharmaceutical preparations can be sterilized and, if desired, bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or one or more other activecompounds, for example, vitamin D₃ and its 1α-hydroxylated metabolites,conjugated estrogens or their equivalents, anti-estrogens, calcitonin,biphosphonates, calcium supplements, cobalamin, pertussis toxin andboron.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solution, as well assuspensions, emulsions, or implants, including suppositories. Parenteraladministration suitably includes subcutaneous, intramuscular, orintravenous injection, nasopharyngeal or mucosal absorption, ortransdermal absorption. Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, lozenges, powders, or capsules. A syrup,elixir, or the like can be used if a sweetened vehicle is desired.

For topical application, suitable nonsprayable viscous, semi-solid orsolid forms can be employed which include a carrier compatible withtopical application and having a dynamic viscosity preferably greaterthan water, for example, mineral oil, almond oil, self-emulsifyingbeeswax, vegetable oil, white soft paraffin, and propylene glycol.Suitable formulations include, but are not limited to, creams,ointments, lotions, solutions, suspensions, emulsions, powders,liniments, salves, aerosols, transdermal patches, etc., which are, ifdesired, sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, demulsifiers, wetting agents, etc. A cream preparation inaccordance with the present invention suitably includes, for example,mixture of water, almond oil, mineral oil and self-emulsifying beeswax;an ointment preparation suitably includes, for example, almond oil andwhite soft paraffin; and a lotion preparation suitably includes, forexample, dry propylene glycol.

Topical preparations of the compound in accordance with the presentinvention useful for the treatment of skin disorders may also includeepithelialization-inducing agents such as retinoids (e.g., vitamin A),chromanols such as vitamin E, β-agonists such as isoproterenol or cyclicadenosine monophosphate (cAMP), anti-inflammatory agents such ascorticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone)and keratoplastic agents such as coal tar or anthralin. Effectiveamounts of such agents are, for example, vitamin A about 0.003 to about0.3% by weight of the composition; vitamin E about 0.1 to about 10%;isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%;hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about 20%;and anthralin about 0.05 to about 2%.

For rectal administration, the compound is formed into a pharmaceuticalcomposition containing a suppository base such as cacao oil or othertriglycerides. To prolong storage life, the composition advantageouslyincludes an antioxidant such as ascorbic acid, butylated hydroxyanisoleor hydroquinone.

For treatment of calcium metabolic disorders, oral administration of thepharmaceutical compositions of the present invention is preferred.Generally, the compound of this invention is dispensed by unit dosageform comprising about 0.5 μg to about 25 μg in a pharmaceuticallyacceptable carrier per unit dosage. The dosage of the compound accordingto this invention generally is about 0.01 to about 1.0 μg/kg/day,preferably about 0.04 to about 0.3 μg/kg/day.

For topical treatment of skin disorders, the dosage of the compound ofthe present invention in a topical composition generally is about 0.01μg to about 50 μg per gram of composition.

For treatment of cancers, the dosage of 1α,24(S)-(OH)₂ D₂ in a locallyapplied composition generally is about 0.01 μg to 100 μg per gramcomposition.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the efficacy of thespecific compound employed, the particular compositions formulated, themode of application, and the particular site and organism being treated.For example, the specific dose for a particular patient depends on theage, body weight, general state of health and sex, on the diet, on thetiming and mode of administration, on the rate of excretion, and onmedicaments used in combination and the severity of the particulardisorder to which the therapy is applied. Dosages for a given host canbe determined using conventional considerations, e.g., by customarycomparison of the differential activities of the subject compounds andof a known agent, such as by means of an appropriate conventionalpharmacological protocol.

In a still further aspect, the compound of the present invention canalso be advantageously used in veterinary compositions, for example,feed compositions for domestic animals to treat or prevent hypocalcemia.Generally, the compound of the present invention is dispensed in animalfeed such that normal consumption of such feed provides the animal about0.01 to about 1.0 μg/kg/day.

The following examples are to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.In the following examples proton nuclear magnetic resonance (¹ H NMR)spectra were recorded with a Bruker AM--400(400 MHz) with aspect 3000Computer in CDCl₃ solutions with CHCl₃ as an internal standard. Chemicalshifts are reported in ppm. Ultraviolet spectra were recorded with aHitachi U-2000 Spectrophotometer and are reported for ethanol solutions.

EXAMPLE 1

Generation, purification and identification of 1α,24(?)-(OH)₂ D₂ inhuman liver cells incubated with 1α-(OH)D₂

Substantially pure 1α-(OH)D₂ was obtained from Bone Care International,Inc. of Madison, Wis. The 1α-(OH)D₂ was cultured for 48 hours with cellsderived from a human hepatoma, Hep 3B, in medium devoid of fetal calfserum using known methods in the art.

Lipid extracts of the combined medium and cells were generated by knownmethods in the art and were subjected to high pressure liquidchromatography (HPLC) on Zorbax-S1L developed withhexane/isopropanol/methanol (91:7:2). The putative 1α,24(?)-(OH)₂ D₂metabolite eluted between the parent 1α-(OH)D₂ and standard 1α,25-(OH)₂D₂ (also obtained from Bone Care International, Inc. of Madison, Wis.).(As used herein, the term "1α,24(?)-(OH)₂ D₂ " is meant to indicate thatthe epimeric form has not been identified.) The 1α,24(?)-(OH)₂ D₂ wasfurther purified by this HPLC system before the metabolite'sidentification was undertaken using mass spectrometry analysis.

The purified metabolite was more polar than the starting material,1α-(OH)D₂ and thus was tentatively concluded to be a dihydroxy vitaminD₂ metabolite. This metabolite also possessed the vitamin D chromophore,indicating retention of the cis-triene system of vitamin D. Since themetabolite was derived from 1α-(OH)D₂, its structure was thus 1α,X-(OH)₂D₂ where "X" indicates the position of the second hydroxyl group.

The trimethylsilyl-derivative of the 1α,X-(OH)₂ D₂ was preparedaccording to known methods in the art and mass spectrometry wasperformed on the TMS-derivative and the native compound. TheTMS-derivative was analyzed by GC-MS, and the identification was mainlyderived from interpretation of the fragmentation pattern of thepyro-metabolite. The molecular ion possessed a m/z of 644 indicating adihydroxy vitamin D₂ with addition of three TMS groups accounting for216 units of additional mass. Since 1α-(OH)D₂ has 3β- and 1α-groups andthe putative metabolite had one additional hydroxyl, all three hydroxylswere thus derivatized. Distinctive fragments were found at m/z 601, 511,421, 331 representing loss of a 43 mass unit of fragment alone or inaddition to one, two or three TMS groups of 90 units each. This patternwas most likely explained by cleavage of the C-24 to C-25 bond loss ofC₃ H₇ accounting for 43 mass units. This represents loss of the C₂₆ -C₂₅-C₂₇ fragment. Furthermore, the mass spectrum lacked the m/z 131fragment characteristic of all 25-hydroxylated vitamin D compounds.

The mass spectrum showed the m/z 513 fragment indicating loss of 131mass units due to A-ring cleavage with loss of C₂ -C₃ -C₄ alsocharacteristic of vitamin D compounds. The mass spectrum also containedm/z 143 which was probably derived from C-24 to C-23 cleavage and a lossof a methyl group. The unusual loss of 43 units indicating C₂₄ -C₂₅fragility coupled with the loss of a fragment due to C₂₃ -C₂₄ cleavageindicated that the extra hydroxyl in 1α,X-(OH)₂ D₂ was at carbon-24.Thus, the structure was identified as 1α,24(?)-(OH)₂ D₂.

The native metabolite was analyzed by direct probe mass spectrometry.This analysis was consistent with a hydroxyl in the 24 position, and wasalso consistent with the GC-MS analysis of the TMS-derivative describedabove. The native metabolite showed the expected molecular ion at m/z428 and a distinctive fragment at m/z 367, indicating the loss of onewater and the C₂₅ -C₂₆ -C₂₇ fragment of 43 mass units.

EXAMPLE 2

Synthesis of 1α,24(S)-dihydroxy vitamin D₂

(22E)-5,7,22-ergostatriene-3β-yl acetate (2)

To a solution of 50 gm (0.13 mol) of ergosterol (1) in 300 ml ofanhydrous pyridine was added 33.3 ml (0.35 mol) of acetic anhydride. Themixture was stirred at room temperature overnight and then 600 ml ofwater was added. The precipitate was filtered and washed three timeswith 200 ml portions of acetonitrile and then air dried to yield 42.0 g(74%) of (2).

22-oxo-5α,8α-(4-phenyl-3.5-dioxo-1,2,4-triazolidine-1.2-diyl)23,24-dinor-6-cholene-3β-ylacetate (4)

To a solution of 33.0 g (0.075 mol) of ergosterol acetate (2) in 1000 mlof chloroform was added 13.2 g (0.075 mol) of4-phenyl-1,2,4-triazoline-3,5-dione. The solution of the thus formed (3)was stirred at room temperature for 30 min. and then 5 ml of pyridinewas added. The solution was cooled to -78° C. and treated at -78° C.with an ozone-oxygen mixture for 2 hours and then thoroughly purged withnitrogen. Then 50 ml of dimethylsulfoxide was added and the mixture waswashed with 300 ml of water, then twice with 200 ml of 2N HCl andfinally 300 ml of water. The organic layer was separated, dried overanhydrous MgSO₄ and concentrated to dryness in vacuo. The residue waspurified on a silica gel column using 30% ethyl acetate in hexane toyield 16.0 g (39%) of the title compound as a foamy solid.

¹ H NMR: (400 MHz; CDCl₃): δppm 0.85 (3H, s, 18-CH₃), 1.10 (3H, s,19-CH₃), 1.15 (3H, d, 21-CH₃), 1.99 (3H, s, 3β-CH₃ CO), 5.45 (1H, m,3α-H), 6.26 (1H, d. 7-H), 6.40 (1H, d, 6-H), 7.42 (5H, m, Ph), 9.58 (1H,d, HCO).

(22E)5,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)cholesta-6,22-diene-24-one-3β-yl acetate (5)

Butyllithium (1.6M solution in hexane 8.94 ml, 0.014 mol) was added to astirred, cooled (0° C.) solution of diisopropylamine (1.45 g, 0.014 mol)in dry tetrahydrofuran (20 ml) under nitrogen. 3-Methylbutan-2-one (1.23g, 0.014 mol) in dry tetrahydrofuran (6 ml) was added dropwise at 0° C.over 15 min. The solution was stirred at 0° C. for 1 hr. more, thencooled to -70° C. and a solution of the aldehyde (4) (6.0 g, 0.011 mol)in dry tetrahydrofuran (60 ml) was added. The temperature was raised to-20° C. and kept at this temperature for 3 hrs. Then glacial acetic acid(20 ml) was added at -20° C. and the solution was brought to roomtemperature. Ether (800 ml) and water (400 ml) were added and theorganic layer was separated and washed with 10% hydrochloric acid (2×300ml), saturated sodium bicarbonate solution (2×300 ml), and water (2×300ml). Concentration gave the crude product (7.5 g) which was dissolved intetrahydrofuran (100 ml) containing 1.5 N-hydrochloric acid (12 ml).After refluxing for 1.5 hrs., the mixture was diluted with ether (600ml), washed with a 5% sodium carbonate solution (2×200 ml) and water(2×200 ml), and dried (anhydrous MgSO₄). Concentration under reducedpressure gave the crude product (7.0 g). Chromatography over silica gel(50% ethyl acetate in hexane) gave the enone (5) 4.0 g (59%).

¹ H NMR: (400 MHz): δppm 0.83 (3H, s. 18-CH₃), 0.99 (3H, s, 19-CH₃),1.09 (6H, dd, 26 and 27-CH₃), 1.12 (3H, d, 21-CH₃), 2.0 (3H, s, 3β-CH₃CO), 2.84 (1H, m, 25-H), 5.45 (1H, m, 3α-H), 6.06 (1H, d, 23-H), 6.24(1H, d, 7-H), 6.39 (1H, d, 6-H), 6.71 (1H, dd, 22-H), 7.42 (5H, m, Ph).

(22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol(6)

The enone (5) (3.5 g, 5.7 mmol) in dry ether (100 ml) was cooled to 0°C. and methylmagnesium bromide (3.0M solution in ether 6.8 ml, 0.02 mol)was added dropwise. After 1 hr. at 0° C., saturated ammonium chloride(100 ml) was added. The organic layer was separated. The aqueous layerwas extracted with ether (2×200 ml). The combined ether phases weredried over anhydrous MgSO₄ and concentrated to dryness in vacuo to yieldthe crude product 3.0 g (90%) of (6).

(22E)-5,7,22-ergostatriene-3β,24-diol (7)

To a solution of 3.0 g (5.1 mmol) of (6) in dry tetrahydrofuran (250 ml)was added 3.6 g (0.09 mol) of lithium aluminum hydride. The mixture washeated under reflux for 3 hrs., cooled with ice water bath and reactionmixture decomposed by the cautious dropwise addition of ice water (5ml). The mixture was filtered and the filtrate was concentrated in vacuoto remove most of the tetrahydrofuran. The residue was dissolved in 200ml of ethyl acetate and washed twice with saturated NaCl solution (2×200ml), dried over anhydrous MgSO₄ and concentrated in vacuo. The residuewas purified on a silica gel column using 30% ethyl acetate in hexane toyield 1.5 g (71%) of (7).

¹ H NMR: (400 MHz, CDCl₃): δppm 0.64 (3H, s, 18-H), 0.88 (6H, dd, 26 and27-CH₃), 0.93 (3H, s, 19-CH₃), 1.06 (3H, d, 21-CH₃), 1.19 (3H, s,28-CH₃), 3.55 (1H, m, 3α-H), 5.36 (1H, d, 7-H), 5.42 (2H, m, 22 and23-H), 5.52 (1H, d, 6-H). UV (ethanol) λ_(max) : 282 nm.

24-hydroxyvitamin D₂ (8)

One gram (2.4 mmol) of (7) was dissolved in 250 ml of ether and benzene(4:1) and irradiated with stirring under nitrogen in a water-cooledquartz immersion well using a Hanovia medium-pressure UV lamp for 2 hrs.The solution was concentrated in vacuo, redissolved in 100 ml of ethanoland heated under reflux overnight. The solution was concentrated todryness in vacuo and the residue was purified on a silica gel columnusing 30% ethyl acetate in hexane to yield 0.55 g (55%) of (8).

¹ H NMR: (400 MHz, CDCl₃): βppm 0.57 (3H, s, 18-CH₃), 0.92 (6H, dd, 26and 27-CH₃), 1.06 (3H, d, 21-CH₃), 1.20 (3H, s, 28-CH₃), 3.93 (1H, m,3-H), 4.79 (1H, m (sharp), 19-H), 5.01 (1H, m, (sharp), 19-H), 5.43 (2H,m, 22 and 23-H), 6.02 (1H, d, 7-H), 6.22 (1H, d, 6-H). UV (ethanol)λ_(max) : 265 nm.

24-hydroxyvitamin D₂ tosylate (9)

To a solution of 0.55 g (1.3 mmol) of (8) dissolved in 5 ml of anhydrouspyridine was added 0.6 g (3.2 mmol) of tosyl chloride. The mixture wasstirred under nitrogen at 5° C. for 20 hrs. The reaction mixture waspoured into 100 ml of cold saturated NaHCO₃ solution and extracted withether (3×100 ml). The combined organic extracts were washed with 5% HClsolution (2×200 ml) saturated sodium bicarbonate solution (2×200 ml) andsaturated NaCl solution (2×200 ml), dried over anhydrous MgSO₄ andconcentrated in vacuo to yield 0.62 g (84%) of (9).

¹ H NMR: (400 MHz, CDCl₃): δppm 0.57 (3H, s, 18-CH₃), 0.92 (6H, dd, 26and 27-CH₃), 1.08 (3H, d, 21-CH₃), 1.24 (3H, s, 28-CH₃), 2.43 (3H, s,CH₃ (tosylate)), 4.69 (1H, m, 3-H), 4.77 (1H, m, (sharp), 19-H), 5.0(1H, m, (sharp), 19-H), 5.42 (2H, m, 22 and 23-H), 6.03 (1-H, d, 7-H),6.25 (1-H, d, 6-H) 7.31 and 7.83 (4H, d, aromatic).

24-hydroxy-3,5-cyclovitamin D₂ (10)

To a solution of 0.6 g (1.06 mmol) of (9) dissolved in 50 ml ofanhydrous methanol was added sodium bicarbonate 4.0 (0.047 mol). Themixture was heated at reflux for 6 hrs. The reaction mixture wasconcentrated in vacuo. Water (100 ml) was added followed by extractionwith ether (2×200 ml). The combined ether extracts were dried overanhydrous MgSO₄ and concentrated to dryness in vacuo to yield 450 mg(100%) of (10) as an oil.

1α,24-dihydroxy-3,5-cyclovitamin D₂ (11)

tert-Butyl hydroperoxide (870 μl (2.61 mmol); 3M in toluene) was addedto a suspension of 73 mg (0.66 mmol) of selenium dioxide in 50 ml ofanhydrous dichloromethane under nitrogen. The mixture was stirred atroom temperature under nitrogen for 3 hrs. Then 0.1 ml of anhydrouspyridine was added followed by a solution of 450 mg (1.06 mmol) of (10)dissolved in 15 ml of anhydrous dichloromethane. The mixture was stirredunder nitrogen at room temperature for 10 min. then 25 ml of 10% NaOHsolution was added and the mixture was extracted with ether (3×100 ml).The combined ether extracts were washed with 10% NaOH solution (2×100ml), water (2×100 ml), saturated sodium chloride solution (2×100 ml),dried over anhydrous MgSO₄ and concentrated to dryness in vacuo. Theresidue was purified on a silica gel column using a mixture of 30% ethylacetate in hexane to yield 110 mg (24%) of (11).

¹ H NMR: (400 MHz, CDCl₃): δppm, 0.55 (3H, s, 18CH₃), 0.90 (6H, dd, 26and 27-CH₃), 1.03 (3H, d, 21-CH₃), 1.19 (3H, s, 28-CH₃), 3.25 (3H, s,--OCH₃), 4.19 (1H, d, 6-H), 4.19 (1H, m, 1-H), 4.92 (2H, d, 7-H), 5.15(1H, m, (sharp), 19-H), 5.2 (1H, m, (sharp), 19-H), 5.42 (2H, m, 22 and23-H).

5,6-cis and 5,6-trans-1α,24-dihydroxy vitamin D₂ (12, 13)

1α,24-dihydroxy-3,5-cyclovitamin D₂ (11) 110 mg (0.25 mmol) wasdissolved in 2.0 ml of dimethylsulfoxide and 1.5 ml of acetic acid andheated at 50° C. under nitrogen for 1 hr. The solution was poured overice and 50 ml of saturated NaHCO₃ solution. The mixture was extractedwith ether (3×100 ml). The combined ether extracts were washed withsaturated NaHCO₃ solution (3×100 ml), water (2×100 ml), saturated NaClsolution (2×200 ml), dried over anhydrous MgSO₄ and concentrated invacuo to yield the crude product 100 mg (93%) of (12) and (13).

5,6-cis-1α,24-dihydroxy vitamin D₂ (12)

To a solution of (12) and (13) in 5 ml of ethyl acetate was added 20 mg(0.2 mmol) of maleic anhydride and the mixture was stirred at 35° C. for24 hrs. under nitrogen. The solution was concentrated to dryness invacuo. The residue was purified on a silica gel column using 50% ethylacetate in hexane to yield 20 mg (22%) of (12).

¹ H NMR: (400 MHz, CDCl₃): δppm 0.57 (3H, s, 18-CH₃), 0.89 (6H, dd, 26and 27-CH₃), 1.04 (3H, d, 21-CH₃), 1.21 (3H, s, 28-CH₃), 4.23 (1H, m,3-H), 4.40 (1H, m, 1-H), 5.0 (1H, m, (sharp), 19-H), 5.33 (1H, m,(sharp), 19-H), 5.44 (2H, m, 22 and 23-H), 6.01 (1H, d, 7-H), 6.37 (1H,d, 6-H). UV (ethanol) λ_(max) : 265 nm.

1α,24 (S) -dihydroxy vitam in D₂ (14)

The 24 epimers of 1α,24-(OH)₂ D₂ were separated by high pressure liquidchromatography, performed on a Waters instrument using a reverse-phaseSupelco C-8 prep. column (25 cm×21.2 mm; particle size 12 μm) with thesolvent system, acetonitrile:water, 60:40, 10 mL/min. The epimers weregiven the designations epimer 1 and epimer 2. Under these conditions theretention time of epimer 1 was 63 min., and the retention time of epimer2 was 71 min. using x-ray crystallography, it was determined that thestereochemistry of epimer 2 was 1α,24(R)-(OH)₂ D₂. The stereochemistryof epimer 1 was therefore known to be 1α,2424 (S)-(OH)₂ D₂

EXAMPLE 3

Identification of the stereochemistry and the biologically derived1α,24(?)-(OH)₂ D₂ metabolite by comparison to the chemically synthesizedepimers, 1α,24(S)-(OH)₂ D₂ and 1α,24(R)-(OH)₂ D₂.

The stereochemistry of the biologically generated metabolite obtained asdescribed in example 1, above, was compared by high pressure liquidchromatography and gas chromatography to the chemically synthesizedepimers obtained as described in example 2, above. Based on thesecomparisons, it was determined that the biologically produced metabolitehas the structure, 1α,24(S)-(OH)₂ D₂. FIG. 3 shows a profile of the highpressure liquid chromatography experiment making this comparison. InFIG. 3, epimer 1 is the chemically synthesized 1α,24(S)-(OH)₂ D₂.

(a) High pressure liquid chromatographic comparisons utilized twodifferent columns and solvent systems. On the reverse-phase columnZorbax-ODS (Dupont Instruments; 3μ; 6.2 mm×8 cm) utilizing the solventsystem, acetonitrile:water, 60:40, 1 ml/min., the biological metaboliteemerged at 14.3 min. and 1α,24(S)-(OH)₂ D₂ ran at 14.2 min.; however,1α,24(R)-(OH)₂ D₂ ran at 15.7 min.

On the straight-phase column Zorbax-SIL (Dupont Instruments; 3μ; 6.2mm×8 cm) utilizing the solvent system, hexane:isopropanol:methanol,94:5:1, 1 ml/min., the biological metabolite emerged at 22.4 min. and1α,24(S)-(OH)₂ D₂ ran at 22.4 min.; however, 1α,24(R)-(OH)₂ D₂ ran at22.8.

(b) With gas chromatography, 1α,24(S)-(OH)₂ D₂ co-migrated with thebiologically generated compound whereas the retention time of1α,24(R)-(OH)₂ D₂ was quite different (Table 1).

                  TABLE 1                                                         ______________________________________                                        Gas Chromatography Retention Times of                                         Pyro-Derivatives Relative to Pyro-1α,25-(OH).sub.2 D.sub.3.             Compound       Relative Retention Time*                                       ______________________________________                                        1α,24(S)--(OH).sub.2 D.sub.2                                                           1.0165                                                         1α,24(R)--(OH).sub.2 D.sub.2                                                           1.0098                                                         Biological Metabolite                                                                        1.0163                                                         ______________________________________                                         *Retention time is expressed relative to an internal standard                 1α,25(OH).sub.2 D.sub.3 where the pyroderivatives are compared.    

EXAMPLE 4

Comparison of the biological activity of 1α,24(S)-(OH)₂ D₂ and1α,24(R)-(OH)₂ D₂.

The biological activity in vitro of chemically synthesized1α,24(S)-(OH)₂ D₂ and 1α,24(R)-(OH)₂ D₂ was measured using a vitaminD-dependent transcriptional activation model system in which a vitamin Dreceptor (VDR)-expressing plasmid pSG5-hVDR1/3 and a plasmid p(CT4)⁴TKGH containing a Growth Hormone (GH)-gene, under the control of avitamin D-responsive element (VDRE) were co-transfected into Greenmonkey kidney, COS-1 cells. DNA's for these two vectors were supplied byDr. Mark Haussler, Department of Biochemistry, University of Arizona,Tucson, Ariz.

Tranfected cells were incubated with vitamin D metabolites and growthhormone production was measured. As shown in Table 2, 1α,24(S)-(OH)₂ D₂has significantly more activity in this system than 1α,24(R)-(OH)₂ D.

                  TABLE 2                                                         ______________________________________                                        Vitamin D Inducible Growth Hormone Production                                 in Transfected COS-1 Cells.                                                                   Vitamin D-Inducible                                                           Growth Hormone Production                                                                     Net                                                                 Total GH  vitamin D-inducible                                      Molar      Production*                                                                             GH-production                                 Inducer    Concentration                                                                            (ng/ml)   (ng/ml)                                       ______________________________________                                        Ethanol                44         0                                           25-OH--D.sub.3                                                                           10.sup.-7   245       201                                                     10.sup.-6  1100      1056                                                     10.sup.-5   775       731                                          1α,25-(OH).sub.2 D.sub.3                                                           .sup. 10.sup.-10                                                                          74        30                                                      10.sup.-9   925       881                                                     10.sup.-8  1475      1441                                          1α,24(S)--(OH).sub.2 D.sub.2                                                       .sup. 5 × 10.sup.-10                                                                425       381                                                     5 × 10.sup.-9                                                                      1350      1306                                                     5 × 10.sup.-8                                                                      1182      1138                                          1α,24(R)--(OH).sub.2 D.sub.2                                                       10.sup.-9   80        36                                                      10.sup.-8  1100      1056                                                     10.sup.-7  1300      1256                                          ______________________________________                                         *Averages of duplicate determinations                                    

EXAMPLE 5

Affinity of 1α,24(S)-(OH)₂ D₂ for the vitamin D receptor.

The affinity of 1α,24(S)-(OH)₂ D₂ for the mammalian vitamin D receptor(VDR) was assessed using a commercially available kit of bovine thymusVDR and standard 1,25-(OH)₂ -D₃ solutions from Incstar (Stillwater,Minn.). Purified 1α,24(S)-(OH)₂ D₂ was quantitated by photodiode arrayspectrophotometry and assayed in the radioreceptor assay. Thehalf-maximal binding of 1α,24(S)-(OH)₂ D₂ was approximately 150 pg/mlwhereas that of 1α,25-(OH)₂ D₂ was 80 pg/ml. Thus, the 1α,24(S)-(OH)₂ D₂had a two-fold lower affinity for bovine thymus VDR than does1α,25-(OH)₂ D₃, indicating that 1α,24(S)-(OH)₂ D₂ had potent biologicalactivity.

EXAMPLE 6

Relative affinities of 1α,24(S)-(OH)₂ D₂ and 1α,24(R)-(OH)₂ D₂ for thevitamin D receptor.

The relative affinities of 1α,24(R)-(OH)₂ D₂ and 1α,24(S)-(OH)₂ D₂ forthe vitamin D receptor (VDR) were assessed using commercially availablereagents of bovine thymus VDR and standard 1α,25-(OH)₂ D₃ solutions fromIncstar (Stillwater, Minn.). The purified 1α,24(R)-(OH)₂ D₂ and1α,24(S)-(OH)₂ D₂ epimers were quantitated by ultraviolet spectroscopy.The concentration of 1α,24(R)-(OH)₂ D₂ required to produce the samedisplacement of ³ H-1α,25-(OH)₂ D₃ tracer from the receptor was 20 to 30times that required for 1α,24(S)-(OH)₂ D₂, as shown in FIG. 4. Thesedata indicate that the activity of the 1α,24(S)-(OH)₂ D₂ epimer issignificantly greater than that of the 1α,24(R)-(OH)₂ D₂ epimer.

EXAMPLE 7

Affinity of 1α,24(S)-(OH)₂ D₂ for the vitamin D serum binding protein.

The affinity of 1α,24(S)-(OH)₂ D₂ for the vitamin D serum bindingprotein (DBP) was assessed using vitamin D deficient rat serum accordingto known methods in the art. The data indicated that the 1α,24(S)-(OH)₂D₂ binding of DBP was at least 1000 times weaker than that for 25-OH-D₃.Given the strong binding of 1α,24(S)-(OH)₂ D₂ for the VDR and weakbinding for the DBP, this compound would tend to be taken up by targetcells, thus possessing a potent biological activity. In addition, theweak binding by the DBP was indicative of more rapid clearance, allowingfor low toxicity.

Thus, the preceding assays demonstrated that the new 1α,24(S)-(OH)₂ D₂exhibited a distinct and unique spectrum of activities-namely, highbiological potency and low toxicity which clearly distinguished thecompound from those of the prior art and from its 24(R) epimer.

EXAMPLE 8

Generation of 1α,24(S)-(OH)₂ D₂ from vitamin D₂ and 24-OH-D₂.

Vitamin D₂ or 24-OH-D₂ was administered (either oral or intraperitonealsupplementation) to vitamin D-deficient rats. Lipid extracts of theplasma were prepared and the metabolites purified by the method of Horstet al. (Horst, A. L., Koszewski, N. J. and Reinhardt, T. A., Biochem.,29:578-82 (1990)) described below for synthesyzing standard biological1α,24-(OH)₂ D₂.

Standard biological 1α,24-(OH)₂ D₂ was synthesized in vitro from24-OH-D₂ by incubating 10 μg of 24-OH-D₂ in flask containing 5 ml of 20%kidney homogenates made from vitamin D-deficient chicks. The product ofthis reaction was isolated by HPLC and identified by mass spectrometry.In the lipid extracts of the plasma from the vitamin D-deficient ratsadministered vitamin D₂ or 24-OH-D₂, one metabolite isolated co-migratedon HPLC with the standard 1α,24-(OH)₂ D₂, indicating that 1α,24-(OH)₂ D₂is a natural metabolite of vitamin D₂. In contrast, comparable ratsadministered vitamin D₃ had no detectable 24-OH-D₃.

EXAMPLE 9

Preferential production of 1α,24(S)-(OH)₂ D₂ with increased substrateconcentrations in vitro.

Hep 3B cells were incubated with 1α-OH-D₂, as described above, at finalconcentrations of 1, 10, or 100 nM (Experiment 1), and 1 or 10 μM(Experiment 2) and 1α,24(S)-(OH)₂ D₂ was extracted and purified. The1α,24(S)-(OH)₂ D₂ and 1α,25-(OH)₂ D₂ metabolites were quantitated byrecovered radiolabel (Experiment 1) or by photodiode arrayspectrophotometry (Experiment 2). As shown in Table 3, the amount of1α,24(S)-(OH)₂ D₂ increased relative to the amount of 1α,25-(OH)₂ D₂ asthe substrate concentration was raised. This indicates that in thissystem 1α,24(S)-(OH)₂ D₂ was the predominant natural active metaboliteof 1α-OH-D₂ at higher substrate concentrations.

                  TABLE 3                                                         ______________________________________                                        EXPER- SUBSTRATE                                                              IMENT  CONCENTRATION PRODUCT FORMED                                           ______________________________________                                                             Ratio of 1α,24(S)--(OH).sub.2 D.sub.2              1      nM            to 1α,25-(OH).sub.2 D.sub.2                        ______________________________________                                                1            1:4                                                              10           1:1                                                             100           1.5:1                                                    ______________________________________                                                             Rate of Production,                                                           pmol per 10.sup.6 cells/day                              2      μM         1α,24(S)--(OH).sub.2 D.sub.2                                                         1α,25-(OH).sub.2 D.sub.2              ______________________________________                                                1            4.9           N.D.*                                              10           59           7.4                                         ______________________________________                                         *N.D. means not detectable                                               

EXAMPLE 10

Production of 1α,24(S)-(OH)₂ D₂ in osteoporotic women administered1α-(OH ₂ D₂.

An increase in the production of 1α,24(S)-(OH)₂ D₂ relative to1α,25-(OH)₂ D₂ has also been observed by the present inventors in humanfemales who received 1α-OH-D₂ as part of an investigation of that drugfor the treatment of osteoporosis. Following either a single dose of 2μg of 1α-OH-D₂ or daily doses of 8 μg/day for one week, blood wascollected and analyzed for the metabolites 1α,24(S)-(OH)₂ D₂ and1α,25-(OH)₂ D₂. Lipid was extracted from the blood, and the metaboliteswere purified by HPLC using standard methods and quantified with theradioreceptor assay produced by Incstar (Stillwater, Minn.). One dayafter a single 2 μg dose, the level of 1α,24(S)-(OH)₂ D₂ wasundetectable with the 1α,25-(OH)₂ D₂ level being approximately 11 pg/ml.In contrast, one day following the last dose of 8 μg, the level of1α,24(S)-(OH)₂ D₂ averaged 9 pg/ml with the 1α,25-(OH)₂ D₂ levelaveraging 30 pg/ml.

EXAMPLE 11

Dose ranging study in postmenopausal osteoporotic women.

Twenty postmenopausal osteoporotic women are enrolled in an open labelstudy. The selected patients have ages between 55 and 75 years, andexhibit L2-L3 vertebral bone mineral density between 0.7 and 1.05 g/cm²,as determined by measurements with a LUNAR Bone Densitometer (LunarCorporation, Madison, Wis.).

On admission to the study, all patients receive instruction on selectinga daily diet containing 400 to 600 mg of calcium. Compliance to thisdiet is verified at weekly intervals by 24-hour food records and byinterviews with each patient.

All patients complete a one-week baseline period, a five-week treatmentperiod, and a one-week post-treatment observation period. During thetreatment period, patients orally self-administer 1α,24(S)-dihydroxyvitamin D₂ at an initial dose of 0.5 μg/day for the first week, and atsuccessively higher doses of 1.0, 2.0, 4.0, and 8.0 μg/day in each ofthe following four weeks. All doses are administered before breakfast.

Blood and urine chemistries are monitored on a weekly basis throughoutthe study. Key blood chemistries include fasting serum levels ofcalcium, phosphorus, osteocalcin, creatinine, and blood urea nitrogen.Key urine chemistries include 24-hour excretion of calcium, phosphorus,and creatinine.

Blood and urine data from this clinical study indicate that thiscompound does not adversely affect kidney function, as determined bycreatinine clearance and blood levels of urea nitrogen; nor does itincrease urinary excretion of hydroxyproline, indicating the absence ofany stimulatory effect on bone resorption. The compound has no effect onany routinely monitored serum parameters, indicating the absence ofadverse metabolic effects.

A positive effect of 1α,24(S)-dihydroxy vitamin D₂ on calciumhomeostasis is evident from modest increases in 24-hour urinary calciumlevels, confirming that the compound increases intestinal calciumabsorption, and from increases in serum osteocalcin levels, indicatingthat the compound stimulates the osteoblasts.

EXAMPLE 12

Preventive treatment of bone mass loss in postmenopausal osteoporoticwomen.

A clinical study is conducted with postmenopausal osteoporoticout-patients having ages between 55 and 75 years. The study involves upto 120 patients randomly divided into three treatment groups andcontinues for 24 to 36 months. Two of the treatment groups receiveconstant dosages of 1α,24(S)-dihydroxy vitamin D₂ (u.i.d.; two differentdose levels at or above 1.0 μg/day) and the other group receives amatching placebo. All patients maintain a normal intake of dietarycalcium (500 to 800 mg/day) and refrain from using calcium supplements.Efficacy is evaluated by pre-and post-treatment comparisons of thepatient groups with regard to (a) total body calcium retention, and (b)radial and spinal bone mineral density as determined by dual-photonabsorptiometry (DPA) or dual-energy x-ray absorptiometry (DEXA). Safetyis evaluated by comparisons of urinary hydroxyproline excretion, serumand urine calcium levels, creatinine clearance, blood urea nitrogen, andother routine determinations.

The results show that patients treated with 1α,24(S)-dihydroxy vitaminD₂ exhibit significantly higher total body calcium, and radial andspinal bone densities relative to patients treated with placebo. Themonitored safety parameters confirm an insignificant incidence ofhypercalcemia or hypercalciuria, or any other metabolic disturbance with1α,24(S)-dihydroxy vitamin D₂ therapy.

EXAMPLE 13

Prophylaxis of postmenopausal bone loss.

A clinical study is conducted with healthy postmenopausal women havingages between 55 and 60 years. The study involves up to 80 patientsrandomly divided into two treatment groups, and continues for 24 to 36months. One treatment group receives a constant dosage of1α,24(S)-dihydroxy vitamin D₂ (u.i.d.; a dose level at or above 1.0μg/day) and the other receives a matching placebo. The study isconducted as indicated in Example 2 above.

The results show that patients treated with 1α,24(S)-dihydroxy vitaminD₂ exhibit reduced losses in total body calcium, radial or spinal bonedensities relative to baseline values. In contrast, patients treatedwith placebo show significant losses in these parameters relative tobaseline values. The monitored safety parameters confirm the safety oflong-term 1α,24(S)-dihydroxy vitamin D₂ administration at this doselevel.

EXAMPLE 14

Management of hypocalcemia and the resultant metabolic bone disease inchronic hemodialysis patients.

A twelve-month, double-blind, placebo-controlled clinical trial isconducted with thirty men and women with renal disease who areundergoing chronic hemodialysis. All patients enter an 8-week controlperiod during which time they receive a maintenance dose of Vitamin D₃(400 IU/day). After this control period, the patients are randomizedinto two treatment groups: one group receives a constant dosage of1α,24(S)-dihydroxy vitamin D₂ (u.i.d.; a dosage greater than 3.0 μg/day)and the other group receives a matching placebo. Both treatment groupsreceive a maintenance dosage of Vitamin D₃, maintain a normal intake ofdietary calcium, and refrain from using calcium supplements. Efficacy isevaluated by pre- and post-treatment comparisons of the two patientgroups with regard to (a) direct measurements of intestinal calciumabsorption, (b) total body calcium retention, (c) radial and spinal bonemineral density, or (d) determinations of serum calcium. Safety isevaluated by regular monitoring of serum calcium.

Analysis of the clinical data show that 1α,24(S)-dihydroxy vitamin D₂significantly increases intestinal calcium absorption, as determined bydirect measurements using a double-isotope technique. Patients treatedwith this compound show normalized serum calcium levels, stable valuesfor total body calcium, and stable radial and spinal bone densitiesrelative to baseline values. In contrast, patients treated with placeboshow frequent hypocalcemia, significant reductions in total body calciumand radial and spinal bone density. An insignificant incidence ofhypercalcemia is observed in the treated group.

EXAMPLE 15

Medicament preparations.

A topical cream is prepared by dissolving 1.0 mg of 1α,24(S)-dihydroxyvitamin D₂ in 1 g of almond oil. To this solution is added 40 gm ofmineral oil and 20 gm of self-emulsifying beeswax. The mixture is heatedto liquify. After the addition of 40 ml hot water, the mixture is mixedwell. The resulting cream contains approximately 10 μg of1α,24(S)-dihydroxy vitamin D₂ per gram of cream.

EXAMPLE 16

An ointment is prepared by dissolving 1.0 mg of 1α,24(S)-dihydroxyvitamin D₂ in 30 g of almond oil. To this solution is added 70 gm ofwhite soft paraffin which had been warmed just enough to be liquified.The ointment is mixed well and allowed to cool. This ointment containsapproximately 10 μg 1α,24(S)-dihydroxy vitamin D₂ per gram of ointment.

EXAMPLE 17

To the ointment of Example 14 is added with thorough mixing 0.5 g ofadenosine and 2.0 g of papaverine base, both dissolved in a minimumquantity of dimethyl sulfoxide. The additional ingredients are presentto the extent of about 0.5 wt. % (adenosine) and 2 wt. % (papaverinebase).

EXAMPLE 18

To the ointment of Example 14 is added with thorough mixing 10,000 U ofVitamin A dissolved in a minimum quantity of vegetable oil. Theresultant ointment contains about 100 U Vitamin A per gram of theointment.

EXAMPLE 19

A dermatological lotion is prepared by dissolving 1.0 mg of1α,24(S)-dihydroxy vitamin D₂ in 100 g of dry propylene glycol. Thelotion is stored in a refrigerator in a brown bottle and contains about10 pg of 1α,24(S)-dihydroxy vitamin D₂ per gram of lotion.

EXAMPLE 20

In 1 g of almond oil is dissolved 0.2 mg of 1α,24-dihydroxy vitamin D₂.To the solution is added 40 g of mineral oil and 20 g ofself-emulsifying beeswax, followed by 40 ml of hot water. The mixture ismixed well to produce a cosmetic cream containing about 2.0 μg of1α,24(S)-dihydroxy vitamin D₂ per gram of cream.

EXAMPLE 21

To a cosmetic cream prepared according to example 18 is added 100 mgadenosine. The cream is mixed well and contains about 0.1 wt. %adenosine.

EXAMPLE 22

An ointment is prepared by dissolving 100 μg of 1α,24(S)-dihydroxyvitamin D₂ in 30 g of almond oil. To the solution so produced is added70 g white soft paraffin which had been warmed just enough to beliquified. The ointment is mixed well and allowed to cool. The ointmentso produced contains about 1.0 μg of 1α,24-dihydroxy vitamin D₂ per gramof ointment.

EXAMPLE 23

To the cosmetic ointment of Example 18 is added with thorough mixing 200U/g Vitamin A dissolved in a minimum amount of vegetable oil.

EXAMPLE 24

A cosmetic lotion is prepared by dissolving 300 μg of 1α,24-dihydroxyvitamin D₂ in 100 g of dry propylene glycol. The lotion is stored in arefrigerator in a brown bottle and contains about 3.0 μg1α,24(S)-dihydroxy vitamin D₂ per gram of lotion.

EXAMPLE 25

Dermatological testing.

Compositions containing 1α,24(S)-dihydroxy vitamin D₂ are evaluated fortherapeutic efficacy of the composition in the topical treatment ofdermatitis (contact and ectopic). The composition evaluated is anointment containing 10 μg of 1α,24-dihydroxy vitamin D₂ per gram ofointment in a petrolatum-almond oil base. The control composition isidentical except that it does not contain the active agent1α,24(S)-dihydroxy vitamin D₂. The patients are treated in anout-patient clinic. They are instructed to use the preparation two timesa day.

The ointment is as far as possible applied to a single lesion, or to anarea of the disease. The ointment and its container are weighed beforethe treatment starts and returned with any unused contents forreweighing at the end of the treatment.

The area of the lesion treated is estimated and recorded, and the lesionis photographed as required, together with suitable "control" lesions.The latter are preferably lesions of similar size and stage ofdevelopment, either in the vicinity of the treated lesion orsymmetrically contralateral. Relevant details of the photographicprocedure are recorded so as to be reproduced when the lesions are nextphotographed (distance, aperture, angle, background, etc.). The ointmentis applied twice daily and preferably left uncovered. The "control"lesions are left untreated, but if this is not possible, the treatmentused on them is noted.

Evaluations of erythema, scaling, and thickness are conducted at weeklyintervals by a physician, with the severity of the lesion rated from 0to 3. The final evaluation is usually carried out at the end of four tosix weeks of treatment. Those lesions treated with 1α,24(S)-(OH)₂ D₂have lower scores than the control lesions. An insignificant incidenceof hypercalcemia is also observed.

EXAMPLE 26

Epidermal cell differentiation and proliferation testing.

Human keratinocytes are cultured according to known modifications of thesystem originally described by Rheinwald and Green (Cell, vol. 6, p. 331(1975)). The 1α,24(S)-dihydroxy vitamin D₂, dissolved in ethanol, isadded to cells to yield a variety of concentrations between 0.05 and 5μg/ml with the ethanol concentration not to exceed 0.5% v/v. Controlcultures are supplemented with ethanol at a final concentration of 0.5%v/v.

Differentiation and proliferation of epidermal cells in culture isexamined by:

1. quantitation of cornified envelopes;

2. quantitation of cell density of cells attached to disks;

3. monitoring transglutaminase activity; or

4. monitoring DNA synthesis by incorporation of ³ H-thymidine.

Cultures incubated with 1α,24(S)-dihydroxy vitamin D₂ have morecornified envelopes, fewer attached cells, higher transglutaminaseactivity, and lower DNA synthesis than control cultures.

EXAMPLE 27

Activity of 1α,24(S)-(OH)₂ D₂ in HL-60 cell differentiation assay.

A dose-response study is conducted with 1α,24(S)-(OH)₂ D₂ in the HL-60cell differentiation assay as described by DeLuca and Ostrom (DeLuca, H.F. and Ostrem, V. K., Prog. Clin. Biol. Res., vol. 259, pp. 41-55(1988)). In this study, 1α,25-(OH)₂ D₃ is used as a positive control andappropriate solvents are used as negative controls. The followingvariables are evaluated: nonspecific acid esterase activity, nitrobluetetrazolium (NBT) reduction, and thymidine incorporation. The resultsshow that 1α,24(S)-(OH)₂ D₂ has potent activity in promotingdifferentiation of HL-60 promyelocytes to monocytes.

EXAMPLE 28

Antiproliferative activity of 1α,24(S)-(OH)₂ D₂ in human cancer celllines.

Dose-response studies are conducted with 1α,24(S)-(OH)₂ D₂ in a batteryof human cancer cell lines. These cell lines include, but are notlimited to, the following: BCA-1 or ZR-75-1 (breast) and COL-1 (colon),as described by Shieh, H. L. et al. Chem. Biol. Interact., vol. 81, pp.35-55 (1982). In this study, appropriate solvents are used as negativecontrols. The results show that 1α,24(S)-(OH)₂ D₂ has potent (andreversible) antiproliferative activity, as judged by inhibition ofthymidine incorporation.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions and omissions, that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

We claim:
 1. A method of inhibiting proliferative activity of coloncancer cells, comprising treating said cancer cells with an effectiveamount of 1α,24(S)-dihydroxyvitamin D₂.
 2. The method of claim 1,wherein said colon cancer cells are COL-1.
 3. The method of claim 1,wherein said inhibiting of proliferative activity is measured byinhibition of thymidine uptake by said colon cancer cells.
 4. The methodof claim 1, wherein said 1α,24(S)-dihydroxy vitamin D₂ is substantiallyfree of its (R) isomer.
 5. The method of claim 1, wherein said treatingstep comprises contacting said colon cancer cells to 1α,24(S)-dihydroxyvitamin D₂.
 6. The method of claim 1, wherein said treating stepcomprises exposing said colon cancer cells to said 1α,24(S)-dihydroxyvitamin D₂ in said amount sufficient to induce differentiation of saidcells to nonmalignant macrophages.
 7. A method of treating colon cancer,comprising administering to a patient suffering therefrom an effectiveamount of 1α,24(S)-dihydroxyvitamin D₂.
 8. The method of claim 7,wherein said amount is sufficient to inhibit proliferative activity ofCOL-1 colon cancer cells.
 9. A method of inhibiting the proliferativeactivity associated with colon cancer, comprising administering to apatient suffering from colon cancer an effective amount of1α,24(S)-dihydroxyvitamin D₂, substantially free of its (R)stereoisomer.